Interpretation of chest fluoroscopy: the risk of misdiagnosing atelectasis as pneumothorax due to greyscale inversion.

Anaesthetists may be required to work in hybrid theatres for procedures using fluoroscopic imaging. Adequate knowledge of fluoroscopic images allows prompt and effective emergency management of complications which arise during procedures. Here, we present a case of severe hypotension and hypoxia occurring shortly after induction of anaesthesia. Atelectasis was mistaken for a pneumothorax due to misinterpretation of fluoroscopic imaging, which demonstrated a dark pleural cavity peripheral to a partially collapsed left lung, leading to an incorrect diagnosis. This case highlights the importance of understanding greyscale inversion in fluoroscopy.

Strategy for Remediation of Polychlorinated Biphenyls-Contaminated Soil Through Redox Management Based on Electronegativity of the Contaminants.

Literature review reveals that Persistent Organic Pollutants (POPs), such as polychlorinated biphenyls (PCBs), are electron deficient compounds due to the presence of highly electronegative groups. Hence, they are more amenable to anaerobic biodegradation rather than oxidative metabolism. However, the studies on PCBs bioremediation are more inclined towards aerobic treatment. Besides, the past studies are mainly centered on screening and application of PCB-degrading microorganisms. In our opinion the degradative capacity is already present in the native microflora, and choice of electron donor is of paramount importance for faster reductive metabolism of PCBs. In this study, the use of methanol as electron donor with cow dung as the general microbial inoculum resulted in high specific rate of degradation (0.0542-0.0637 /day) for high-chlorinated biphenyls. The % removal of PCBs ranged between 67.7 and 71.7%. It may be the first study on the application of methanol as a cheap electron donor for PCBs biodegradation without bioaugmentation with specifically selected microorganisms.

Dynamic ultrasound-guided short-axis needle tip navigation technique vs. landmark technique for difficult saphenous vein access in children: a randomised study.

Dynamic ultrasound-guided short-axis needle tip navigation is a novel technique for vascular access. After venipuncture, the needle and catheter are further advanced within the vessel lumen under real-time ultrasound guidance with constant visualisation of the needle tip in the short-axis view. This can minimise the risk of transfixing the cannulated vessel. We compared two techniques for non-visible saphenous vein cannulation under general anaesthesia in children weighing ≥ 3 kg and less than four years of age: dynamic ultrasound-guided short-axis needle tip navigation technique (ultrasound group) vs. landmark technique. Venous cannulation was performed by three experienced anaesthetists. The primary outcome measure was first-attempt success rate. Success rate within 10 min was a secondary outcome. A total of 102 patients were randomly allocated to either the ultrasound group or the landmark group. First-attempt success rate was 90% in the ultrasound group compared with 51% in the landmark group, p<0.001, difference 39%, 95% confidence interval (CI) of the difference 23-55%. Success rate within 10 min was 92% in the ultrasound group compared with 63% in the landmark group, p = 0.001, difference 29%, 95%CI of the difference 14-45%. We conclude that, when performed by experienced anaesthetists, the dynamic ultrasound-guided short-axis needle tip navigation technique improved non-visible saphenous vein cannulation in children compared with the landmark technique.

Assays to Monitor Pexophagy in Yeast.

Pexophagy is a selective autophagy process that degrades damaged and/or superfluous peroxisomes in the yeast vacuole or in mammalian lysosomes. The molecular mechanisms of pexophagy are well studied in yeast. Peroxisomes can be rapidly induced by oleate in the budding yeast, Saccharomyces cerevisiae, and by oleate or methanol in the methylotrophic yeast, Pichia pastoris. A number of peroxisomal matrix enzymes, such as 3-ketoacyl CoA thiolase (thiolase) and alcohol oxidase (AOX), are upregulated correspondingly to meet metabolic demands of the cells. Removal of these peroxisome-inducing carbon sources creates conditions wherein peroxisomes are superfluous and results in pexophagy and the degradation of these peroxisomal matrix enzymes. In this chapter, we discuss different assays to monitor pexophagy in yeast. These assays rely on tracking the localization of the BFP-SKL protein (a peroxisomally targeted version of the blue fluorescent protein) by microscopy, biochemical analysis of the degradation of peroxisomal matrix proteins, thiolase and AOX, and/or measuring the reduction of AOX activity during pexophagy.

Microincision cataract surgery combined with vitrectomy: a case series.

AIM: The objective of this study was to present the results of combined phacovitrectomy using 1.8 mm microincision cataract surgery (MICS) with special emphasis on the anterior segment complications in this group. METHODS: Retrospective, single-centre case series involving consecutive patients undergoing phacovitrectomy in a single centre in the United Kingdom during a 6-month period. RESULTS: A total of 52 eyes underwent combined MICS and pars plana vitrectomy. Intraoperative complications included posterior capsule rupture (n=2), minor iris trauma during phacoemulsification (n=1), iatrogenic retinal tears (n=2), and entry site break (n=1). Postoperatively two cases had significant inflammation, one of which resulted in 360° posterior synaechiea, iris bombe, and raised intraocular pressure. Other complications included mild posterior synaechiae (n=2), posterior capsular opacification (n=3), cystoid macular oedema (n=1), and hyphaema (n=1), which spontaneously resolved. There were no cases of intraocular lens decentration. Two patients who underwent surgery for retinal detachment repair subsequently redetached. Among those having surgery for macular hole, non-closure was seen in one patient and one patient developed a retinal detachment. CONCLUSION: In conclusion, sub-2 mm MICS is a safe and effective technique in dealing with vitreoretinal disorders necessitating cataract surgery at the same time.

Serum uric acid levels and its association with age-related macular degeneration (ARMD).

UNLABELLED: To investigate the possible association between serum uric acid levels, serum C-Reactive Protein (CRP), and age-related macular degeneration (ARMD). A total 232 patients of the eye department at Hospital Tuanku Ja'afar, Negeri Sembilan, Malaysia were recruited over 9 weeks. Participants were divided into ARMD (Non-Neovascular ARMD, and Neovascular ARMD) and control groups. 107 participants with non-neovascular ARMD, 6 with neovascular ARMD, and 119 controls participated in the study. The control patients had a similar average Serum Uric Acid level to the average of all patients with ARMD (P = 0.617). CONTROL GROUP: mean 299.19 micromol/l +/- std dev. 89.847 micromol/l. ARMD group: mean 302.53 micromol/l +/- std dev. 80.794 micromol/l. The average serum uric acid levels were higher in patients with neovascular ARMD (median = 397 mean +/- std dev = 389.67 +/- 38 micromol/l) than in the non-neovascular ARMD group (288.5 micromol/l, 297.86 +/- 80.26 micromol/l), and control group (295.5 micromol/l, 299.19 +/- 89.95 micromol/l). Comparing the standardised serum uric acid levels in the control group (Median = 0.5) against the two ARMD groups separately, there was no significant difference to the non-neovascular group (P = 0.448) but there was a difference significant to the neovascular ARMD group (P = 0.044). The neovascular and non-neovascular ARMD groups had median CRP value of 0.25 mg/l and were not significantly different. There is no association between serum uric acid levels and ARMD as a whole. There is potentially an association between serum uric acid and neovascular ARMD, an association needs to be established further. There is no association between serum CRP and ARMD.

4-aminopyridine-induced contracture in frog ventricle is due to calcium released from intracellular stores.

The aim of the study is to demonstrate the presence of intracellular calcium store in frog ventricle based on contractures induced by 4-aminopyridine in calcium-free media. Frog-ventricular strips were subjected to field stimulation at 0.2 Hz and the force of contraction was recorded after stabilization. The preparation was then kept quiescent for some time in solutions with different sodium concentrations, containing 0 or 1 mmol/L calcium. Caffeine, 4-aminopyridine (4-AP), or tetraethylammonium chloride was then added. Frog skeletal muscle preparations were used as positive controls for the caffeine experiments. Frog ventricular preparations did not develop contractures (sustained contractions) in the presence of caffeine (25 mmol/L), while frog skeletal muscle preparations developed caffeine-induced contractures. However, 4-AP (16 mmol/L) was able to induce contractures in quiescent frog ventricular preparations, even when they were superfused with calcium-free solution. 4-AP contractures in frog ventricle were seen in the presence of nifedipine also. Amplitude of 4-AP evoked contractures in frog ventricle were much larger in low sodium (30 mmol/L) and sodium-free (sodium substituted by lithium) solutions than in normal sodium solution, suggesting that the route of extrusion of the cytosolic calcium (released from intracellular stores by 4-AP) is the sodium calcium exchanger, which gets reversed in low sodium solutions. Tetraethylammonium chloride (TEA) was not able to induce contractures in frog ventricle suggesting that the contracture evoked by 4-AP is not due to its potassium channel blocking effect. In quiescent frog skeletal muscle preparations, caffeine as well as 4-AP induced contractures in calcium-free solutions. We therefore conclude that there is a caffeine-insensitive, 4-AP sensitive intracellular calcium store in the frog ventricle.

Force-frequency relation in frog-ventricle is dependent on the direction of sodium/calcium exchange in diastole.

AIM: Force of contraction increases with stimulus-frequency in mammalian and amphibian hearts under control conditions. Here, we have analysed the mechanism of the force-frequency relation (FFR) in frog-ventricle. METHODS: Circular strips of frog-ventricle were subjected to field-stimulation with frequencies in the range 0.03-0.2 Hz and force recorded on a chart-recorder. In another protocol, varying rest-periods were imposed while the preparation beat steadily at 0.2 Hz and the effect of rest on post-rest beat amplitude was noted. RESULTS: Under control conditions, a positive FFR and a rest-induced decay of contraction amplitude (RID) were seen in the frequency range 0.03-0.2 Hz. With cadmium, nifedipine, nickel (40 micromol L(-1)), ryanodine and adrenaline (all drugs at 10 micromol L(-1) concentration, except nickel), the positive FFR and RID seen under control conditions persisted. When the bathing solution contained ouabain (10 micromol L(-1)) or low external sodium (40 mmol L(-1)), or high external calcium (5 mmol L(-1)), the FFR turned negative in the frequency range stated above and there were rest-induced potentiations (RIP). CONCLUSION: When the conditions favour a net leak of calcium in diastole from intracellular stores via the calcium-extrusive mode of sodium-calcium exchanger (NCX), FFR is positive. An increase in frequency lessens the diastolic interval and therefore the diastolic calcium leak, thereby augmenting force. On the other hand, interventions which favour the calcium-acquisitive mode of NCX during diastole, changed the pattern of RID to RIP and converted FFR from positive to negative. With net diastolic calcium uptake, there is better store-filling and therefore higher force at lower frequencies.

Self-destruction in the line of duty.

Three cellular processes, microautophagy, macroautophagy, and the cytoplasm-to-vacuole (Cvt) pathway, are involved in the cargo delivery from the cytosol to the vacuole or lysosome. Recent findings have identified Cvt19 at the receptor for specific cargo binding in the Cvt pathway.

Identification of a novel human peroxisomal 2,4-dienoyl-CoA reductase related protein using the M13 phage protein VI phage display technology.

Recently, we reported the successful use of the gVI-cDNA phage display technology to clone cDNAs coding for novel peroxisomal enzymes by affinity selection using immobilized antisera directed against peroxisomal subfractions (Fransen, M.; Van Veldhoven, P.P.; Subramani, S. Biochem. J., 1999, 340, 561-568). To identify other unknown peroxisomal enzymes, we further exploited this promising approach. Here we report the isolation and cloning of another novel human cDNA encoding a protein ending in the tripeptide AKL, a C-terminal peroxisomal targeting signal (PTS1). Primary structure analysis revealed that this molecule shared the highest sequence similarity to members of the 2,4-dienoyl-CoA reductase (DCR) family. However, functional analysis indicated that a recombinantly expressed version of the novel protein did not possess DCR activity with either 2-trans,4-trans-hexadienoyl-CoA or 2-trans,4-trans-decadienoyl-CoA as a substrate. The recombinant protein interacted with HsPex5p, the human PTS1-binding protein. Binding was competitively inhibited by a PTS1-containing peptide and was abolished when the last amino acid of the PTS1 signal was deleted. Transfection of mammalian cells with gene fusions between green fluorescent protein (GFP) and the human cDNA confirmed a peroxisomal localization and, therefore, the functionality of the PTS1. These results further demonstrate the suitability of the gVI-cDNA phage display technology for cDNA expression cloning using an antibody as a probe.

Genomic organization, chromosomal localization and tissue specific expression of the murine Pxmp2 gene encoding the 22 kDa peroxisomal membrane protein (Pmp22).

Peroxisomes are subcellular organelles with important functions in lipid metabolism that are found in virtually all eucaryotic cells. The peroxisomal membrane contains a number of integral and peripheral membrane proteins involved in the import of peroxisomal matrix proteins and the transport of metabolites across the membrane. The most abundant peroxisomal membrane protein (Pmp) in rat peroxisomes is Pmp22, a 22 kDa protein of unknown function that is encoded by the Pxmp2 gene. To investigate the function of the Pxmp2 gene, we have initiated mouse knockout studies. The expression level of the Pxmp2 mRNA in mice was investigated by Northern blot analysis. Pxmp2 RNA was shown to be differentially expressed with highest expression levels in liver, kidney and in heart tissue. Comparison with other peroxisomal marker genes revealed that the expression of Pxmp2, Pmp70 (Pxmp1) and catalase was regulated independently. Using 5' and 3' RACE we have cloned the full-length cDNA of murine Pxmp2 which comprises 863 nucleotides and have isolated a genomic clone containing the entire murine Pxmp2. We have analyzed the complete intron/exon structure of the Pxmp2 gene which contains five exons spanning about 11 kb on the genomic clone. All intron/exon splice junctions conform to the GT/AG rule. Sequence analysis of the Pxmp2 5' flanking region revealed that it was devoid of a TATA box, but characteristic promoter elements were identified within 250 base pairs upstream of the transcriptional start site. Using a mouse/hamster radiation hybrid panel, Pxmp2 was localized on mouse chromosome 5 at 59 cM.

The human peroxisomal targeting signal receptor, Pex5p, is translocated into the peroxisomal matrix and recycled to the cytosol.

Peroxisomal targeting signals (PTSs) are recognized by predominantly cytosolic receptors, Pex5p and Pex7p. The fate of these PTS receptors following their interactions on the peroxisomal membrane with components of docking and putative translocation complexes is unknown. Using both novel and multiple experimental approaches, we show that human Pex5p does not just bind cargo and deliver it to the peroxisome membrane, but participates in multiple rounds of entry into the peroxisome matrix and export to the cytosol independent of the PTS2 import pathway. This unusual shuttling mechanism for the PTS1 receptor distinguishes protein import into peroxisomes from that into most other organelles, with the exception of the nucleus.

Pichia pastoris Pex14p, a phosphorylated peroxisomal membrane protein, is part of a PTS-receptor docking complex and interacts with many peroxins.

The peroxisomal protein import machinery plays a central role in the assembly of this organelle in all eukaryotes. Genes encoding components of this machinery, termed peroxins or Pex proteins, have been isolated and characterized in several yeast species and in mammals, including humans. Here we report on one of these components, Pex14p, from the methylotrophic yeast Pichia pastoris. Work in other organisms has shown that Pex14p is located on the cytoplasmic surface of the peroxisomal membrane and binds peroxisomal targeting signal (PTS) receptors carrying proteins bound for the peroxisomal matrix, results that have led to the hypothesis that Pex14p is a receptor-docking protein. P. pastoris Pex14p (PpPex14p) behaves like an integral membrane protein, with its C-terminus exposed on the cytosolic side of the peroxisomal membrane. PpPex14p complexes with many peroxins, including Pex3p (Snyder et al., 1999b), Pex5p, Pex7p, Pex13p, Pex17p, itself, and a previously unreported peroxin, Pex8p. A portion of Pex14p is phosphorylated, but both phosphorylated and unphosphorylated forms of Pex14p interact with several peroxins. The interactions between Pex14p and other peroxins provide clues regarding the function of Pex14p in peroxisomal protein import.

Quantitative analysis of peroxisomal protein import in vitro.

Protein import into the peroxisome matrix is mediated by peroxisome-targeting signals (PTSs). We have developed a novel, quantitative, in vitro assay for measuring peroxisomal import of PTS1-containing proteins. This enzyme-linked immunosorbent assay-based system utilizes semi-intact human A431 cells or fibroblasts and a biotinylated version of the PTS1-containing import substrate, luciferase. We show that biotinylated luciferase accumulated in peroxisomes in a time- and temperature-dependent fashion, in a reaction stimulated by exogenously added ATP, cytosol, and zinc. No import was detected in fibroblasts from a human patient belonging to complementation group 2, who suffered from the fatal peroxisomal disorder Zellweger syndrome and lacked a functional PTS1 receptor, Pex5p. Also, the reaction was significantly inhibited by antibodies to the zinc-finger protein, Pex2p. Several lines of evidence demonstrate that biotinylated luciferase was imported into the lumen of bona fide peroxisomes. (a) Biochemical fractionation of cells after the import reaction showed a time-dependent accumulation of the import substrate within intracellular organelles. (b) Confocal fluorescence microscopy indicated that imported biotinylated luciferase colocalized with the peroxisomal protein PMP70. (c) Visualization of the imported biotinylated luciferase by indirect fluorescence or indirect immunofluorescence required disruption of the peroxisomal membrane, indicating true import rather than binding to the outside of the organelle.

Import of peroxisomal matrix and membrane proteins.

This review summarizes the progress made in our understanding of peroxisome biogenesis in the last few years, during which the functional roles of many of the 23 peroxins (proteins involved in peroxisomal protein import and peroxisome biogenesis) have become clearer. Previous reviews in the field have focussed on the metabolic functions of peroxisomes, aspects of import/biogenesis the role of peroxins in human disease, and involvement of the endoplasmic reticulum in peroxisome membrane biogenesis as well as the degradation of this organelle. This review refers to some of the earlier work for the sake of introduction and continuity but deals primarily with the more recent progress. The principal areas of progress are the identification of new peroxins, definition of protein-protein interactions among peroxins leading to the recognition of complexes involved in peroxisomal protein import, insight into the biogenesis of peroxisomal membrane proteins, and, of most importance, the elucidation of the role of many conserved peroxins in human disease. Given the rapid progress in the field, this review also highlights some of the unanswered questions that remain to be tackled.

The peroxin Pex19p interacts with multiple, integral membrane proteins at the peroxisomal membrane.

Pex19p is a protein required for the early stages of peroxisome biogenesis, but its precise function and site of action are unknown. We tested the interaction between Pex19p and all known Pichia pastoris Pex proteins by the yeast two-hybrid assay. Pex19p interacted with six of seven known integral peroxisomal membrane proteins (iPMPs), and these interactions were confirmed by coimmunoprecipitation. The interactions were not reduced upon inhibition of new protein synthesis, suggesting that they occur with preexisting, and not newly synthesized, pools of iPMPs. By mapping the domains in six iPMPs that interact with Pex19p and the iPMP sequences responsible for targeting to the peroxisome membrane (mPTSs), we found the majority of these sites do not overlap. Coimmunoprecipitation of Pex19p from fractions that contain peroxisomes or cytosol revealed that the interactions between predominantly cytosolic Pex19p and the iPMPs occur in the organelle pellet that contains peroxisomes. These data, taken together, suggest that Pex19p may have a chaperone-like role at the peroxisome membrane and that it is not the receptor for targeting of iPMPs to the peroxisome.

The CUP1 promoter of Saccharomyces cerevisiae is inducible by copper in Pichia pastoris.

We report the construction of a Pichia pastoris integrating vector which contains the inducible CUP1 promoter from Saccharomyces cerevisiae. We show that the promoter is indeed inducible by copper when used in P. pastoris and that the level of induction is dependent on the amount of copper in the medium.